Project Summary Ubiquitylation describes the process by which the small protein ubiquitin is covalently attached to other proteins. Most, if not all, cellular processes are regulated in some way by ubiquitylation. Consequently, defects in components of the ubiquitin system are associated with several human diseases, most notably cancer and neurodegeneration. Over the past decade we have gained tremendous insight into the molecular components and processes that conjugate ubiquitin to substrate proteins as well as the subsequent degradation by the 26S proteasome. This knowledge stems primarily from studies of protein degradation. Protein ubiquitylation has therefore become a synonym for a signal that induces proteolysis. Surprisingly, comparative proteome-wide ubiquitin profiling experiments using proteasome inhibition estimate that only about 60% of ubiquitylated proteins are efficiently degraded by the proteasome, implying that protein ubiquitylation has widespread signaling functions independent of mediating proteolysis. Despite the importance of ubiquitylation in biology and human health, and the apparent importance of non- proteolytic functions of ubiquitylation, molecular concepts governing ubiquitin signaling are understudied and our understanding remains rudimentary. However, detailed insight into processing of the diverse ubiquitin signals will be important for basic biomedical research and development of therapeutics targeting the ubiquitin system. Key questions are: Why are some ubiquitylated proteins degraded and others are not? How can ubiquitylation directly affect protein activity? What is the role of different ubiquitin chain topologies and posttranslational modifications on ubiquitin? Over the past years we have developed a highly defined system that allows us to study these questions in great detail. This system is focused on the cullin-RING ubiquitin ligase complex SCFMet30, which connects metabolic or heavy metal stress to cell cycle regulation. SCFMet30 modifies a number of substrates with the canonical degradation signal, the lysine-48 (K48) linked ubiquitin chain. Interestingly, while some substrates behave as expected and are targeted for degradation by the 26S proteasome, other substrates are regulated in a proteolysis-independent manner. This proposal builds on a plethora of tools we have developed during past funding periods of this grant, which will allow us to analyze biochemistry and physiology of ubiquitin signaling by addressing the following questions: (1) how is the critical change of the ubiquitin chain topology from K48 to K11 achieved and how does it induce transcription factor activation; (2) how does ubiquitylation trigger protein complex remodeling through recruitment of Cdc48/p97 complexes, (3) what are roles and mechanisms of the recently identified herterodimeric SCF complex with the two essential F-box proteins Met30 and Cdc4 in restricting centromeric histone H3 incorporation; (4) how do F-box proteins sense metabolic states and heavy metals; and (5) how does phosphorylated ubiquitin modulate ubiquitin signaling. Ubiquitylation affects many important cellular processes and has been linked to a number of human diseases including cancer, neurodegeneration, and retroviral infection. A contribution of proteolysis- dependent and independent mechanisms is evident. It will be important to understand the molecular concepts that govern ubiquitin signaling to design diagnostic tools and treatment strategies. This proposal aims to achieve detailed mechanistic insight into signaling through ubiquitin and to define the concepts of proteolytic as well as regulatory ubiquitylation pathways.